Liquid chromatography injection system

ABSTRACT

A method and apparatus for quickly and efficiently injecting variable size fluid samples into the solvent flow stream of a high pressure liquid chromatography system. The injection apparatus includes a laterally moveable valve member that is formed with a pair of parallel transverse passages and is initially positioned such that one of the passages is aligned with the solvent flow stream. The apparatus further includes means for receiving and supporting a syringe so that the end of the needle is disposed within the valve at the other transverse valve passage, a selectively engageable sealing member for sealing the syringe needle and valve once the syringe is in position, and means for simultaneously moving the syringe, sealing member, and valve to a position such that said other transverse valve passage is in said flow stream enabling a sample within the syringe to be injected into the flow stream without depressurization of the system or substantial interruption of the flow stream.

United States Patent [1 1 Spaans et al.

[4 1 May 20, 1975 1 LIQUID CHROMATOGRAPHY INJECTION SYSTEM [75] Inventors: Johannes Spaans, Schipluiden;

Adrianus Onderwater, Delft, both of Netherlands [73] Assignee: Packard Becker BV, Netherlands [22] Filed: Oct. 5, 1973 [21] Appl. No.: 403,791

[52] US. Cl. 210/31 C; 210/198 C [51] Int. Cl B01d 15/08 [58] Field of Search... 55/67, 386; 210/31 C, 198 C OTHER PUBLICATIONS Text: Gas Phase Chromatography by Rudolf Kaiser,

Vol. 1, page 97 relied on, published by Butterworths Inc., Washington, DC, 1963.

Primary Examiner John Adee Attorney, Agent, or Firm-Wolfe, Hubbard, Leydig, Voit & Osann, Ltd.

571 ABSTRACT A method and apparatus for quickly and efficiently injecting variable size fluid samples into the solvent flow stream of a high pressure liquid chromatography system. The injection apparatus includes a laterally moveable valve member that is formed with a pair of parallel transverse passages and is initially positioned such that one of the passages is aligned with the solvent flow stream. The apparatus further includes means for receiving and supporting a syringe so that the end of the needle is disposed within the valve at the other transverse valve passage, a selectively engageable sealing member for sealing the syringe needle and valve once the syringe is in position, and means for simultaneously moving the syringe, sealing member, and valve to a position such that said other transverse valve passage is in said flow stream enabling a sample within the syringe to be injected into the flow stream without depressurization of the system or substantial interruption of the flow stream.

12 Claims, 6 Drawing Figures LIQUID CHROMATOGRAPHY INJECTION SYSTEM DESCRIPTION OF THE INVENTION The present invention relates generally to liquid chromatography, and more particularly, to an improved apparatus and method for introducing small fluid samples into high pressure liquid chromatography systems.

In recent years, several different types of injection methods have been proposed for introducing samples into liquid chromatography systems. Each of these prior methods, however, has had certain drawbacks. In one method, for example, a selected quantity of the sample is inserted into the column at atmospheric pressure, following which the system is pressurized to force a carrier fluid through the chromagraphic column. This method suffers from the disadvantage that the column has to be depressurized before each sample is introduced. It also results in inevitable loss in measuring precision from such extreme pressure variations.

Another sample injection means has involved the use of a syringe which is inserted through a membrane type of port, called a septum. The syringe/septum technique, unfortunately, is not well suited for pressures above 200 p.s.i.g. because the septum generally does not have the requisite strength to withstand such pressures. A further problem with the septum is that its lifetime is relatively short due to the repeated needle perforation and its solubility frequently is in the sensitivity range with the liquid chromagraphic carrier solvents.

Sample introduction also has been effected by means of an external sample loop that is selectively connected into the solvent flow stream. Such sample loop methods, however, require a relatively large minimum sample size, such as microliters. This technique further generally necessitates a multifunctional valve loop prior to introduction of the sample into the system thereby increasing the complexity of the apparatus and enhancing the likelihood of leakage.

In addition, samples have been introduced by means of high pressure valves. Such prior sample valve techniques have been relatively robust and complex, and accordingly rather costly, and, as in the case of the fixed loop method, have had the significant limitation of permitting only the introduction of a fixed sample volume. In liquid chromatography systems, it is highly desirable to be able to vary the sample volume. If the quantity of sample is limited, the use of relatively small samples is necessitated. A fixed volume system simply does not have the versatility for effective use in all situations.

Accordingly, it is an object of the present invention to provide an injection method and apparatus for liquid chromatography systems in which a variable sample quantity may be introduced into the system without reducing the pressure of the system and without detectably interrupting the solvent flow stream.

Another object is to provide an injection apparatus as characterized above in which a relatively small sample may be quickly and easily introduced into the flow stream of the system without adversely affecting the precision measuring capabilities of the system.

A further object is to provide sample injection means of the above kindwhich is relatively simple in construction, and thus, is economical to manufacture, use and maintain.

Other objects and advantages of the invention will become apparent upon reading the foregoing detailed description and upon reference to the drawings, in which: I

FIG. I the longitudinal partially diagrammatic section of an injection apparatus embodying the present invention;

FIG. 2 is a longitudinal section similar to FIG. I, but showing a sample holding syringe inserted therein at a ready position;

FIG. 3 is an enlarged section of a portion of the apparatus shown in FIG. I, but with the valve member of the apparatus moved to a position to permit introduction of the sample into the solvent flow stream from the syringe;

FIG. 4 the diagrammatic flow chart of a liquid chromagraphic system in which the injection apparatus of the present invention is incorporated;

FIG. 5 shows a typical recording trace produced by the chromatography system shown in FIG. 3 after a sample has passed through the system;

FIG. 6 is a chart showing the spreading effect of a sample as a function of the solvent flow.

While the invention is susceptible of various modifications and alternative constructions, a certain illustrated embodiment has been shown in the drawings and will be described below in detail. It should be understood, however, that there is no intention to limit the invention to the particular form disclosed herein, but on the contrary, the intention is to cover all modifications, alternative constructions and equivalents falling within the spirit and scope of the invention.

Referring more particularly to FIG. 4 of the drawings, there is shown a flow diagram of a typical liquid chromatography system 10. The basic components of the system 10 include a solvent reservoir 11 and a pump 12 for directing a continuous flow stream of solvent from the reservoir through a line 14 and column 15 at relatively high linear speeds. The column 15 customarily has a relatively small bore of not more than a few millimeters in diameter and is packed with fine solids such as silica gel, alumina, or porous glass beads. The pressure of the solvent in the column 15 preferably is in the range of ISO to 250 atmospheres. As the solvent flow stream is pumped through the column 15, a solution of the sample to be tested is introduced into the fast-moving stream by an injector l6 and is rapidly carried through the column.

As is well known in the liquid chromatography field,

the different components of the sample solution will separate into respective bands while passing through the column due to their interaction with the material contained therein. As the separated sample components elute from the column, they pass through one or more detectors 18 of a known type which produce signals that are recorded as deviations from a baseline. As is well known in the art, the peak position along the curve relative to the starting point denotes the particular component once its elution characteristics have been determined for the given set of operating conditions. With proper calibration, the height or area of the peak is a measurement of the amount of the component present in the sample. It is conventional practice to locate both the injector and the detector in close positions relative to the ends of the column so as to reduce so called dead volume in which elutional spreading of the sample occurs, with the resulting undesirable widening of the bands.

The manner in which the sample is injected into the flow stream of such a high pressure liquid chromatography system, as indicated previously, can have a significant effect on the output accuracy of the system. If the sample is introduced too slowly, or unevenly, into the solvent stream, this will have a tendency to widen or otherwise adversely affect the bands measured by the detector. Substantial interruptions of the flow stream or variances in pressure can also affect the accuracy of the output. Likewise, the size of the sample required for dependable results may vary significantly depending upon the concentration of the sample that is injected into the system.

In accordance with the present invention, the injector 16 is adapted to introduce variable sample quantities into the high pressure liquid chromatography system without reducing the pressure of the solvent stream or substantially interrupting its flow. As best shown in FIGS.1-3, the injector is supported within a panel 19, such as that of a cabinet containing the system, by brackets 20 and 21 each secured to the panel 19 by bolts 22 or other appropriate fasteners. A valve housing 24 is supported behind the panel 19 by the bracket 21, and the line 14 through which the solvent flow stream passes is connected into opposite sides of the valve housing 24. The valve housing 24 is formed with transversely aligned ports 25a and 25b of substantially the same diameter as the internal diameter of the line 14, preferably about 0.25 millimeters, to permit the passage of the stream through the housing.

A valve member is mounted within the housing 24 for relative lateral sliding movement. To facilitate such movement,'bearing sleeves 31 are provided within the housing 24 about the valve member. The valve member 30 in this case is cylindrically shaped and formed with a pair of parallel radial passages 34 and 35. The valve member 30, as shown in FIGS. 1 and 2, is positioned within the housing 24 with the passage 35 aligned with the housing ports 25a, 25b so that solvent stream flows from the line 14, through housing port 25b, the valve passage 35, and the housing port 25a. Extending laterally from the other radial passage 34, to the left as shown in the drawings, is a longitudinal port 36. The outer end of the valve member 30 is formed with an internal counter-bored section 38 having a tapered or conical shaped end 39 that joins the longitudinal port 36. A pair of O-rings 40 are provided between the valve member 30 and housing 24 on opposite sides of the flow stream to prevent leakage of the pressurized solvent along the valve member.

To facilitate moving the valve member 30 laterally, a transfer sleeve 41 is connected by set screws 42 to the outwardly extending end of the valve member 30. The transfer sleeve 41 extends through an aperture in the bracket 21 and has an enlarged cylindrical portion 43 slidably received within a cylindrical opening in the bracket 20. The enlarged transfer sleeve portion 43 is formed with internal threads which operatively engage external threads formed on an extension 44 of a rotatable knob or handle 45 supported coaxially within the transfer sleeve 41 and bracket 20. To retain the knob 45 from lateral movement with respect to the bracket 20, the end of the bracket 20 is formed with a depending flange 46 that is received within a channel 48 formed in the knob. Suitable means, such as the key connection (not shown) between the bracket 20 and the transfer sleeve 41, serves to prevent rotational movement between the two parts while permitting relative longitudinal movement. Thus, by reason of the threaded connection between the knob 45 and the transfer sleeve 41, it will be seen that by rotating the knob 45, the transfer sleeve 41 and the valve member 30 may be laterally advanced and retracted relative to the brackets 20, 21 and valve housing 24.

Mounted concentrically within the transfer sleeve 41 and knob 45 is a syringe guide member 50. The guide member 50 is formed with a reduced diameter portion 51 that is slidably received within the counter-bore 38 of the valve member 30. A narrower sleeve 52 extends from the reduced diameter portion 51 of the guide member 50 further into the valve member counterbore 38. A seal member 54, made of plastic material such as that commonly sold under the trade name KEL- F, surrounds the guide member sleeve 52 and is formed with an outer shape substantially in conformity with the tapered internal configuration of the valve member counter-bore section 38.

In order for the guide member to receive a syringe 58 which contains a sample for testing, the guide member 50 is formed with an internal cylindrical bore 59 having a conical or tapered end 60 and a small longitudinal port 61 extending axially from the apex of the tapered end 60. The longitudinal port 61 is axially aligned with the valve member longitudinal port 54, and the sealing member 54 is formed with a similar axially aligned port. When the syringe 58 is inserted into the guide member bore 59, the tapered end 60 will guide the syringe needle 62 into the longitudinal port 61. The syringe needle may be pushed through the sealing member 54 until the end is located at the valve member transverse passage 34, as shown in FIG. 2.

To enable the sealing member to form a firm and tight seal about the syringe needle 62 after it has been positioned within the guide member 50 and valve member 30, the guide member 50 is longitudinally adjustable with respect to the transverse sleeve 41 and valve member 30 so as to compress the sealing member 54 about the needle. To this end, the guide member 50 is formed with an externally threaded shoulder 64 that operably engages an internal threaded portion of the transfer sleeve 41. By turning an outer knob or handle 65 of the guide member 50, the guide member may be advanced inwardly into the valve member 30 to force the sleeve 54 into the tapered counter-bore end 39, which in turn will tend to compress the sealing member 54 into tight contact with a syringe needle 62.

To introduce a sample into the flow stream of the liquid chromatography system 10, the operation of the injector 16 is as follows. The desired quantity of the liquid to be tested, the amount of which will depend upon the concentration and availability of the sample, is drawn into the syringe 58. The syringe then is inserted into the bore 59 of the guide member 50 and the syringe needle 62 is positioned into the longitudinal port 61 and pushed through the seal 54 until the end of the needle reaches the transverse passage 34. Preferably, the end of the needle 62 should be substantially flush with the side of the port 34 so as not to interfere with the fluid flow which subsequently will pass through the port 34, as will become apparent. The guide member knob 65 may then be rotated to axially advance the guide member 50 into the valve counter-bore 38 to force the sealing member 54 into tight sealing engagement around the needle 62. After the seal has been tightened, the knob 45 may be rotated to laterally move the transfer sleeve 41, the valve member 30, guide member 50, the syringe 58, and sealing member 54 to the right, as viewed in FIGS. 1-3, to a position where the transverse passage 35 is removed from the flow path and the passage 34 replaced therein. Since the end of the syringe needle 62 remains located at the passage 34 duringsuch lateral translational movement, depression of the syringe plunger will result in quick and direct injection of the sample into the solvent stream which is then passing through the line 14, and ports 25b, and port 25a, as shown in FIG. 3. It will be appreciated that due to the high velocity of the solvent stream through the valve passage 34, the sample is quickly drawn into the solvent flow path and back diffusion into the longitudinal valve port 61 is prevented.

After injection has been completed, the syringe plunger may be withdrawn to draw solvent from the flow stream into the syringe. Due to the force in which the solvent is moving when it enters the syringe, the syringe is thoroughly cleansed as it is being refilled. After the syringe has been filled with solvent from the flow stream, rotation of the knob 65 in the opposite direction will laterally move the transfer sleeve 41, valve member 30, guide member 50, and sealing member 54 to the left to withdraw the transverse passage 34 and the syringe 58 from the flow stream and reconnect the passage 35. The sealing member 54 then may be released from the needle 62 by reverse rotation of the knob 65, which moves the guide member 50 away from the seal and permits the syringe to be withdrawn from the injector apparatus.

The injection system of the present invention has been found to efficiently and rapidly introduce samples into solvent flow streams at pressures between 15025O atmospheres without adversely affecting the output results of the detector. FIG. 5, for example, shows a recorder tracing produced by an injection of a l microliter sample at 180 atmospheres. It can be seen that no substantial baseline disturbances are attributable to the injection and that relatively narrow band output results were achieved. FIG. 6 further demonstrates the minimum band spreading characteristics which result due to an increased solvent flow through a system. It can be seen that while the flow is increased by more than 40 times, from about one-half to 24 microliters per secend, the band spreading increases less than twice, from about 3 to 5% microliters.

From the foregoing, it will be appreciated that the method of the present invention provides a simple and efficient means for introducing variable size fluid samples into a high pressure liquid chromatography flow stream. Basically, the method, as has been shown, involves the steps of positioning the injector valve member so that one of two transverse passages is in alignment with the solvent flow stream, drawing a desired volume of the sample fluid into a syringe, inserting the syringe into the valve member with the outer end thereof disposed at the second transverse passage of the valve member, applying a tight seal between the syringe needle and the valve member, and moving the valve member and syringe to a position whereby the second passage and the syringe needle outlet located therein is brought into the solvent flow stream so that the solvent passes through the second passage, and injecting the sample from the syringe into the flow stream. Introduction of the sample into the flow stream by this method is achieved without reducing the pressure of the system and without detectably interrupting the flow of the solvent stream.

What is claimed is:

l. A sample injection apparatus for use 'in a liquid chromatography system in which a continuous high pressure, high velocity solvent stream is directed through a column comprising, in combination, a valve housing, a valve member mounted for relative lateral sliding movement within said housing, said valve member being formed with a pair of passages extending transversely through said member, said valve member being positionable such that one of said transverse passages is in alignment with said solvent stream so that said stream flows through said one passage, a sample holding means for selectively drawing in and ejecting variable quantities of a sample, said sample holding means having an outlet at one end thereof and being removably positionable within said valve member such that said outlet is disposed at the other of said transverse valve passages, and means for laterally moving said valve member with said sample holding means positioned therein from a position where said one valve passage is aligned with said solvent stream to a position where said other valve passage is aligned with said solvent stream and said solvent flows through said other passage.

2. The injection apparatus of claim 1 including selectively engageable and releasable sealing means for sealing said sample holding means with respect to said valve member.

3. The injection apparatus of claim 2 wherein said sample holding means is a syringe having a needle, a sample containing portion, and means for drawing in and ejecting a sample from said containing portion, said valve sealing means being engageable with said needle, and said moving means simultaneously moves said valve member, syringe, and sealing means without relative movement therebetween.

4. The injection apparatus of claim 1 in which said valve housing is formed with ports to permit passage of said solvent stream, and said moving means moves said valve member between positions wherein said valve transverse passages are alternately aligned with said housing ports.

5. The injection apparatus of claim 1 in which said valve member includes a coaxially disposed transfer sleeve having a threaded portion, a rotatably mounted handle means having a threaded portion in operative engagement with said threaded transfer sleeve whereby rotation of said handle means causes lateral movement of said transfer sleeve and valve member relative to said housing.

6. The sample injection apparatus of claim 3 including a guide member mounted concentrically within said transfer sleeve for relative lateral movement, said guide member having an end portion received within the end of said valve member, a sealing member disposed within said valve member in surrounding relation to said guide member end portion, said guide member having a central bore for receiving the sample containing portion of said syringe, said guide member, sealing member, and valve member each being formed with concentric axial ports which communicate from said guide member central bore to said other transverse valve passage for receiving the needle of a syringe positioned within said guide member, and means for moving said guide member relative to said transfer sleeve and valve member to cause said guide member to tightly compress said sealing member about the needle of a syringe positioned within said guid member.

7. The apparatus of claim 6 in which said guide member moving means includes an externally threaded shoulder on said guide member that is in threaded engagement with an internally threaded portion of said transfer sleeve whereby rotation of said guide member moves said guide member relative to said sleeve.

8. The apparatus of claim 7 in which said valve is formed with a conical shaped bore for receiving said sealing member, and said sealing member has a complementary shaped conical end that is engageable with said valve bore upon lateral advancing movement of said guide member relative to said transfer sleeve and valve member.

9. The injection apparatus of claim 8 in which said guide member has a handle portion at its outermost end for rotating said guide member relative to said transfer sleeve.

10. A method of introducing a liquid sample into a fluid flow stream of a high pressure liquid chromatography system of a type which has a syringe receiving valve with a pair of transverse passages comprising the steps of drawing into a syringe a desired volume of the liquid sample, positioning said valve so that one of said transverse passages is in the fluid flow stream and fluid flows through said passage, inserting the syringe into the valve member with the outlet end of said syringe disposed at the other of said transverse passages, and moving said valve and syringe to a position whereby said other passage is in the fluid flow stream so that solvent passes through said other passage, and ejecting the sample from the syringe into the flow stream.

11. The method of claim 10 including forming a seal between said syringe and valve prior to moving said valve, and after said seal is formed moving said syringe, valve, and seal without relative movement therebetween.

12. The method of claim 11 including drawing into said syringe a quantity of fluid from said flow stream after said sample has been injected, moving said valve, seal and syringe to a position whereby said other valve passage is removed from said flow stream and said one valve passage is located in said flow stream, releasing the seal about said syringe, and removing said syringe from said valve. 

1. A SAMPLE INJECTION APPARATUS FOR USE IN A LIQUID CHROMATOGRAPHY SYSTEM IN WHICH A CONTINUOUS HIGH PRESSURE, HIGH VELOCITY SOLVENT STREAM IS DIRECTED THROUGH A COLUMN COMPRISING, IN COMBINATION, A VALVE HOUSING, A VALVE MEMBER MOUNTED FOR RELATIVE LATERAL SLIDING MOVEMENT WITHIN SAID HOUSING, SAID VALVE MEMBER BEING FORMED WITH A PAIR OF PASSAGES EXTENDING TRANSVERSELY THROUGH SAID MEMBER, SAID VALVE MEMBER BEING POSITIONABLE SUCH THAT ONE OF SAID TRANSVERSE PASSAGES IS IN ALIGNMENT WITH SAID SOLVENT STREAM SO THAT SAID STREAM FLOWS THROUGH SAID ONE PASSAGE, A SIMPLE HOLDING MEANS FOR SELECTIVELY DRAWING IN AND EJECTING VARIABLE QUANTITIES OF A SAMPLE, SAID SAMPLE HOLDING MEANS HAVING AN OUTLET AT ONE END THEREOF AND BEING REMOVABLY POSITIONABLE WITHIN SAID VALVE MEMBER SUCH THAT SAID OUTLET IS DISPOSED AT THE OTHER OF SAID TRANSVERSE VALVE PASSAGES, AND MEANS FOR LATERALLY MOVING SAID VALVE MEMBER WITH SAID SAMPLE HOLDING MEANS POSITIONED THEREIN FROM A POSITION WHERE SAID ONE VALVE PASSAGE IS ALIGNED WITH SAID SOLVENT STREAM TO A POSITION WHERE SAID OTHER VALVE PASSAGE IS ALIGNED WITH SAID SOLVENT STREAM AND SAID SOLVENT FLOWS THROUGH SAID OTHER PASSAGE.
 2. The injection apparatus of claim 1 including selectively engageable and releasable sealing means for sealing said sample holding means with respect to said valve member.
 3. The injection apparatus of claim 2 wherein said sample holding means is a syringe having a needle, a sample containing portion, and means for drawing in and ejecting a sample from said containing portion, said valve sealing means being engageable with said needle, and said moving means simultaneously moves said valve member, syringe, and sealing means without relative movement therebetween.
 4. The injection apparatus of claim 1 in which said valve housing is formed with ports to permit passage of said solvent stream, and said moving means moves said valve member between positions wherein said valve transverse passages are alternately aligned with said housing ports.
 5. The injection apparatus of claim 1 in which said valve member includes a coaxially disposed transfer sleeve having a threaded portion, a rotatably mounted handle means having a threaded portion in operative engagement with said threaded transfer sleeve whereby rotation of said handle means causes lateral movement of said transfer sleeve and valve member relative to said housing.
 6. The sample injection apparatus of claim 3 including a guide member mounted concentrically within said transfer sleeve for relative lateral movement, said guide member having an end portion received within the end of said valve member, a sealing member disposed within said valve member in surrounding relation to said guide member end portion, said guide member having a central bore for receiving the sample containing portion of said syringe, said guide member, sealing member, and valve member each being formed with concentric axial ports which communicate from said guide member central bore to said other transverse valve passage for receiving the needle of a syringe positioned within said guide member, and means for moving said guide member relative to said transfer sleeve and valve member to cause said guide member to tightly compress said sealing member about the needle of a syringe positioned within said guid member.
 7. The apparatus of claim 6 in which said guide member moving means includes an externally threaded shoulder on said guide member that is in threaded engagement with an internally threaded portion of said transfer sleeve whereby rotation of said guide member moves said guide member relative to said sleeve.
 8. The apparatus of claim 7 in which said valve is formed with a conical shaped bore for receiving said sealing member, and said sealing member has a complementary shaped conical end that is engageable with said valve bore upon lateral advancing movement of said guide member relative to said transfer sleeve and valve member.
 9. The injection apparatus of claim 8 in which said guide member has a handle portion at its outermost end for rotating said guide member relative to said transfer sleeve.
 10. A method of introducing a liquid sample into a fluid flow stream of a high pressure liquid chromatography system of a type which has a syringe receiving valve with a pair of transverse passages comprising the steps of drawing into a syringe a desired volume of the liquid sample, positioning said valve so that one of said transverse passages is in the fluid flow stream and fluid flows through said passage, inserting the syringe into the valve member with the outlet end of said syringe disposed at the other of said transverse passages, and moving said valve and syringe to a position whereby said other passage is in the fluid flow stream so that solvent passEs through said other passage, and ejecting the sample from the syringe into the flow stream.
 11. The method of claim 10 including forming a seal between said syringe and valve prior to moving said valve, and after said seal is formed moving said syringe, valve, and seal without relative movement therebetween.
 12. The method of claim 11 including drawing into said syringe a quantity of fluid from said flow stream after said sample has been injected, moving said valve, seal and syringe to a position whereby said other valve passage is removed from said flow stream and said one valve passage is located in said flow stream, releasing the seal about said syringe, and removing said syringe from said valve. 